"Genomic studies of chimeric mitochondria in cybrids from Solanaceae" This research will be conducted primarily in Argentina at Universidad Nacional de Cuyo in collaboration with Dr. Marma Virginia Sanchez Puerta, as an extension of R01 GM070612-4. The study of mitochondrial genome evolution, dynamics, uptake of foreign DNA, and interactions with the nuclear genome is essential to a deep understanding of the eukaryotic cell. Mitochondrial genomes provide an excellent system to increase our knowledge of genome rearrangements, chimeric genes, nuclear- cytoplasmic incompatibilities, and horizontal gene transfer (HGT). Plant mitochondria are unique in their propensity to acquire genes by HGT. The most pervasive example of HGT in eukaryotes involves the cox1 intron, which encodes a putative homing endonuclease that increases the frequency of the intron's fixation via horizontal transfer. We propose to study aspects of the interactions between two distinct mitochondrial genomes that recombine in a cybrid plant obtained by protoplast fusion experiments. During cybrid formation, the two mitochondrial parental types and their genomes (only one containing the cox1 intron) will fuse, resulting in a hybrid mitochondrial genome. We will establish some 20 cybrid lines derived from somatic crosses between cox1 intron-containing and -lacking species in order to address the following two aims: First, we will test the hypothesis that the cox1 intron encodes a functional homing endonuclease in plants, assess rates of intron colonization, and measure lengths of exonic coconversion tracts that accompany intron insertion. Second, we will sequence and analyze the entire mitochondrial genomes of up to 20 cybrid lines. This will serve as an experimental model for recapitulating the natural process of HGT in plant mitochondria and will provide a rich picture of the process and pattern of mitochondrial genome recombination at various levels. Maintenance of an intact, functioning mitochondrial genome is critical for survival. Given the many molecular parallels between plants and animals, this project will be valuable for the development of both animal gene therapy and plant genetic engineering. PUBLIC HEALTH RELEVANCE: Genome rearrangements, nucleotide substitutions, and the introduction of foreign DNA shape the mitochondrial genomes of eukaryotes and often have severe consequences for human health, as evidenced by several important mitochondrially-inherited diseases. In addition, the key experimental organism of this study (tobacco) is widely used as an expression system for biopharmaceutical production of vaccines, antibiotics, and a number of therapeutic proteins. Understanding the molecular mechanisms of mitochondrial DNA evolution is therefore of considerable importance to human health and disease.